1. Field of the Invention
This invention relates to a method of detecting fluorescent materials and also an apparatus for their detection. More particularly, the invention relates to a method and apparatus for detecting fluorescent materials at high sensitivity in liquid chromatography by exciting fluorescent materials to produce light by chemical reaction with chemical reagents.
2. Description of the Prior Art
Liquid chromatography is widely used for separating and quantitatively analyzing one or several kinds of materials in a mixture of various components. When the material or materials to be separated and quantitatively analyzed have absorptions in the ultraviolet or visible wavelength region, an absorptions spectrophotometer is usually used as the detection means in liquid chromatography. However, when a higher sensitivity is required, for example, in the case where the amount of the aforesaid material or materials to be analyzed in the sample mixture is very small, a fluorescence detector is employed as the detection means in liquid chromatography.
A conventional fluorescence detector for liquid chromatography is composed of a flow cell through which a solution containing separated fluorescent material is passed, a light source for exciting the fluorescent material, and a light-receptive detection means for perceiving and multiplying the light (fluorescence) generated (Instrumental liquid chromatography p. 81, ed by N. A. Paris, Elsevier Scientific Publishing Comp. 1976). In the conventional fluorescence detector, undesirable phenomena, such as the stray light phenomenon wherein a part of light from the light source, for exciting the fluorescent materials, enters the light-receptive detector and a deviation of the light from the light source induces a deviation in the fluorescence. Thus, an attempt to increase the sensitivity of the detector leads to, at the same time, an increase in the noise level and it is difficult to increase the signal to noise ratio (S/N). In such a fluorescent detector, there is a limitation in the increment of the detection sensitivity and thus the detector is insufficient for the detection of a very minute amount of fluorescent materials.
On the other hand, a means for increasing the sensitivity, has been developed in which without using a flow cell for reducing stray light, special light (strong laser light is applied onto a droplet of the eluate from the column for liquid chromatography to excite the fluorescent materials, but the precision is low and the cost of the apparatus is very high. Therefore, the development of a method wherein fluorescent materials can be detected with high sensitivity and high precision using an inexpensive apparatus has been desired.